Endothelial cells line the luminal surface of the vascular bed and are thought to play an active role in the specific proteolytic breakdown of locally deposited fibrin, Todd, J. Pathol. Bacteriol., 78, 281 (1959); Astrup, in Progress in Chemical Fibrinolysis and Thrombolysis, Davidson et al. eds., vol. 3, pp. 1-57, Raven Press, New York (1978). The potential of endothelium to initiate and control this process is emphasized by its capacity to synthesize and release plasminogen activators (PAs), Loskutoff et al., Proc. Natl. Acad. Sci. (USA), 74, 3903 (1977); Shepro et al., Thromb. Res., 18, 609 (1980); Moscatelli et al., Cell, 20, 343 (1980); Laug, Thromb. Haemostasis, 45, 219 (1981); Booyse et al., Thromb. Res., 24, 495 (1981), including both tissue-type and urokinase-type molecules, Levin et al., J. Cell Biol., 94, 631 (1982); Loskutoff et al., Blood, 62, 62 (1983). Endothelial cells can also produce inhibitors of fibrinolysis, Loskutoff et al., Proc. Natl. Acad. Sci. (USA), 74, 3903 (1977); Levin et al., Thromb. Res., 15, 869 (1979); Loskutoff et al., J. Biol. Chem., 256, 4142 (1981); Dosne et al., Thromb. Res., 12, 377 (1978); Emeis et al., Biochem. Biophys. Res. Commun., 110, 392 (1983); Loskutoff et al., Proc. Natl. Acad. Sci. (USA), 80, 2956 (1983); Levin, Proc. Natl. Acad. Sci. (USA), 80, 6804 (1983).
Although these inhibitors probably serve important regulatory roles in controlling the fibrinolytic system of the vascular wall, little is known about their specificity, mode of action, or biochemical nature. The conclusion that these inhibitors are actually synthesized by endothelial cells is obscured somewhat by recent reports that cultured cells can bind and internalize protease inhibitors from serum-containing culture medium, Cohen, J. Clin. Invest., 52, 2793 (1973); Pastan et al., Cell, 12, 609 (1977); Rohrlich et al., J. Cell Physiol., 109, 1 (1981); McPherson et al., J. Biol. Chem., 256, 11330 (1981).
The possibility of producing relatively unlimited amounts of tissue-type plasminogen activator (t-PA) by recombinant DNA technology as described in British patent application GB 2,119,804 A, published Nov. 23, 1983, has generated much interest, both clinically and commercially. The conversion of the relatively inactive molecule into an extremely efficient thrombolytic agent by fibrin itself, suggests that t-PA can exist as an active enzyme only when localized to the fibrin-platelet thrombus itself. Thus, t-PA is considered to be a much more specific thrombolytic agent than urokinase-type plasminogen activator and streptokinase.
The interactions between t-PA and fibrin have raised the argument that natural inhibitors of t-PA are not necessary to regulate this system; i.e., regulation is achieved through the formation/dissolution of fibrin, and, thus, do not exist. It is clear that the existence of such inhibitors in human blood would complicate attempts to design a specific, efficient, and safe thrombolytic program based upon natural and genetically engineered t-PA. At the very least, calculations such as those of dose, treatment time and efficacy of treatment would be difficult to predict and/or monitor. This problem would be especially acute if inhibitor levels varied from individual to individual.
The existence of specific inhibitors of t-PA in plasma is a matter of some dispute, Collen, Thromb. Haemostas., 43, 77 (1980). In fact, it has been reported, Korninger et al., Thromb. Haemostas., 46, 662 (1981), that the activity of t-PA added to plasma had an in vitro half-life of 90 minutes as compared to an in vivo half-life of 2 minutes, Korninger et al., Thromb. Haemostas, 46, 658 (1981). Based upon these observations, those authors concluded that t-PA inhibition by plasma was physiologically unimportant.
That conclusion has recently been challenged in Kruithof et al., Prog. in Fibrinolysis, 6, 362 (1983). In Chmielewska et al., Thromb. Res., 31, 427 (1983), direct evidence was recently reported for the existence of a rapid inhibitor of t-PA in plasma. In all cases, this anti-t-PA activity was detected in the plasma of patients with or at risk to develop thrombotic problems; i.e., the very individuals most likely to receive t-PA therapy. This finding may account for the failure of Korninger et al., Thromb. Haemostas., 46, 662 (1981), to detect such an activity since they only examined the plasma of "normal" individuals. These reports on t-PA inhibitors represent little more than qualitative descriptions of an "activity" detected in the blood of some individuals.
Recently, an antifibrinolytic agent in cultured bovine endothelial cells was detected, Loskutoff et al., Proc. Natl. Acad. Sci. (USA), 80, 2956 (1983). This inhibitor is a major endothelial cell product and is an inhibitor of plasiminogen activator since it can neutralize the activity of both fibrin-independent (urokinase-type) and fibrin-dependent (tissue-type) plasminogen activators (PAs). The observation that human platelets contain an immunologically similar inhibitor, Erickson et al., Haemostasis, 14 (1), 65 (1984) and J. Clin. Invest. 74, 1465 (1984), that is released by them in response to physiologically relevant stimuli, e.g., thrombin, and in parallel with other platelet proteins, e.g., Platelet Factor 4, emphasizes the potential importance of this inhibitor in human biology. Antiserum to the plasminogen activator inhibitor (PAT) from bovine aortic endothelial cells (BAEs) has been employed to show that the human endothelial PAI as well as that from plasma, serum and platelets are related, i.e., immunologically similar. Erickson et al., Proc. Natl. Acad. Sci. USA, 82, 8710 (1985).
The inhibitor found by Loskutoff et al., Proc. Natl. Acad. Sci. (USA), 80, 2956 (1983), was purified from bovine aortic endothelial cell conditioned media by a combination of concanavalin A affinity chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and was shown to be a single chain glycoprotein of a molecular weight of 50,000 daltons, having an isoelectric point of 4.5-5 [van Mourik et al., J. Biol. Chem. 259, 14914 (1984)].
Recent evidence indicates that there are three immunologically distinct plasminogen activator inhibitors (PAIs). The first is that discussed above that is derived primarily from endothelial cells. The second, reported by Astedt et al., Thromb. Haemostasis, 53, 122 (1985) was isolated from placenta. The third, reported by Scott et al., J. Biol. Chem., 260 7029 (1985) is protease nexin.
The endothelial cell type PAI differs, in addition to immunologically, from placental PAI and protease nexin in that it inhibits both single chain and two chain tissue-type plasminogen activator (t-PA) as well as urokinase-type plasminogen activator (u-PA), while protease nexin and the placental PAI exhibit substantially no t-PA inhibition at physiological concentrations. Those latter two inhibitors do inhibit u-PA activity at physiological concentrations. Still further, endothelial PAI exhibits beta-mobility when analyzed by agarose zone electrophoresis while the other two PAIs do not. In addition the endothelial cell PAI is stable to low pH values (e.g. pH 3) and SDS (0.1%), while the other two inhibitors are rapidly inactivated by either of these treatments. [van Mourik et al., J. Biol. Chem., 259, 14914 (1984)].
The results discussed hereinafter illustrate that the endothelial cell type PAT exhibiting beta-mobility is also present in human placental extracts as is the placenta PAI reported by Astedt et al., Thromb. Haemostasis, 53., 122 (1985). Since two types of PAI are obtainable, from placenta, the human PAI hereinbefore referred to as of endothelial cell origin will usually be referred to as beta-PAT or endothelial PAI, or endothelial cell type PAT while the PAI first isolated from placenta is referred to as placental-type PAI or placental PAI.